GSM6022332: Ldi_WT_EV_1; Leishmania infantum; OTHER
Leishmania small EVs were isolated and purified as we previously described (Atayde et al., 2019; Douanne et al., 2020a). Briefly, 1 L of Leishmania parasites (2.5–5.0 x 107 parasites/mL) was grown in drug-free M199 at 25°C (supplemented with 10% FBS and 5 μg/mL of haemin at pH 7.0) and left to divide in 10 non-ventilated 75 cm2 culture flasks (Corning, USA) until they reached late-log phase. Next, parasites were washed twice in PBS and resuspended in 5 mL RPMI-1640 medium (no FBS, no phenol red) (Life Technologies) at a final concentration of 2.5–5.0 x 10^8 parasites/mL, in non-ventilated 25 cm2 culture flasks (Corning, USA) at 37°C. Parasites were stimulated for 4 h at 37°C to maximize the release of EVs in the medium (Atayde et al., 2019; Douanne et al., 2020a). The viability of parasites was evaluated by propidium-iodide (PI) staining before and after incubation at 37°C. Only cultures with a viability > 95.0% were submitted to subsequent EV-purification steps. After the 4-h incubation, samples were centrifuged 10 min at 3,000 g to eliminate parasites and 10 min at 8,500 g to clear out debris, followed by two subsequent filtrations using 0.45 and 0.22 μm syringe filters. EVs were then recovered by a 1 h ultracentrifugation at 100,000 g and resuspended in EVs buffer (137 mM NaCl, 20 mM Hepes, pH 7.5). EV proteins were dosed using the Micro BCA Protein Assay Kit (Pierce Biotechnology, USA) and size and concentration measurements of the purified EV samples were performed with a ZetaView Nanoparticle tracking analyzer (Particle Metrix, USA). DNA was extracted using the DNeasy Blood and Tissue Kit (QIAGEN, USA) from purified EVs, following the manufacturer's instructions, to eliminate external DNA. Dried DNA pellets were resuspended in ddH2O and further concentrated using a Savant DNA SpeedVac Concentrator (Thermo Electron Corporation, USA). Purified DNAs were fluorometrically quantified using the Quant-iT PicoGreen dsDNA Assay Kit (Thermo Fisher Scientific, USA) in a Take 3 platform (BioTek, USA). DNA was stored at -20°C until use in subsequent next generation sequencing or PCR experiments. Extracellular vesicle DNA libraries were constructed using the KAPA HyperPrep kit following the manufacturer's protocol without fragmentation; the final library fragment size was 250-400 bp. Sequencing was performed using an Illumina NovaSeq 6000 sequencer in a 50 bp pair-end configuration. An average 29-fold and 32-fold genome coverage was achieved for the EV DNA samples from the WT and the Sb-resistant mutant, respectively.